Summary Medical treatments for cancer or other non-malignant conditions can cause permanent infertility. The only fertility preservation option available to prepubertal male patients who are not producing sperm is experimental testicular tissue freezing. Prepubertal boys do have spermatogonial stem cells (SSCs) in their testes that have the potential to produce sperm. With this in mind, academic centers around the world are cryopreserving testicular tissues for boys in anticipation that those tissues can be used in the future to restore fertility. Although methods to produce sperm and live offspring from immature frozen tissues have been developed in mice, translation to efficient and safe methods to produce sperm from those tissues in humans has not been achieved. We will use our rhesus macaque model of cancer survivorship to test next generation technologies that might be used to protect endogenous SSCs from gonadotoxic therapies or produce sperm and offspring from cryopreserved, prepubertal testicular tissues. In addition, each patient who preserves testicular tissues at the Fertility Preservation Program in Pittsburgh (https://fertilitypreservationpittsburgh.org/) donates a portion of their tissue to research, which will enable us to extend the studies on macaques to human. SSC transplantation is an established approach to regenerate spermatogenesis after gonadotoxic treatment, but there are limitations to this method. This application will test three alternative approaches that may circumvent some or all of these limitations. Aim 1 will test the hypothesis that co-administration of modulation of granulocyte colony stimulating factor or fibroblast growth factor signaling at or around the time of gonadotoxic treatment will enhance survival of endogenous SSCs and recovery of spermatogenesis. Aim 2 will build on our recent demonstration that cryopreserved testicular tissue from prepubertal Rhesus macaques could be autologously grafted under the back skin or scrotal skin of the same macaque or immunotolerant mice and matured to produce sperm and a healthy baby. Graft recipients in those studies were peripubertal and castrated. Since young fertility preservation patients will not be castrated and may not have tissues re- implanted until adulthood, Aim 2 will confirm that immature testicular tissues can be matured in pubertal or adult animals with intact testes. Immature human testicular tissues will also be grafted and matured in mouse and monkey hosts. The limitations of the grafting approaches are the risk of reintroducing cancer cells if the graft is done in human or exposure to xenotropic viruses if the graft is done in an animal. To circumvent these issues, Aim 3 will utilize a testicular tissue organ culture, developed by Ogawa and colleagues, to mature prepubertal testicular tissues ex vivo. This approach has not been replicated in mice or translated to other species. We propose to replicate, modify and improve the Ogawa technique and compare to other in vitro gametogenesis platforms in mice. Finally, we will determine whether prepubertal monkey or human testicular tissues can be matured to produce sperm efficiently in any of these in vitro gametogenesis platforms.